Abstract

Using a systematic, whole-genome analysis of enhancer activity of human-specific endogenous retroviral inserts (hsERVs), we identified an element, hsERVPRODH, that acts as a tissue-specific enhancer for the PRODH gene, which is required for proper CNS functioning. PRODH is one of the candidate genes for susceptibility to schizophrenia and other neurological disorders. It codes for a proline dehydrogenase enzyme, which catalyses the first step of proline catabolism and most likely is involved in neuromediator synthesis in the CNS. We investigated the mechanisms that regulate hsERVPRODH enhancer activity. We showed that the hsERVPRODH enhancer and the internal CpG island of PRODH synergistically activate its promoter. The enhancer activity of hsERVPRODH is regulated by methylation, and in an undermethylated state it can up-regulate PRODH expression in the hippocampus. The mechanism of hsERVPRODH enhancer activity involves the binding of the transcription factor SOX2, whch is preferentially expressed in hippocampus. We propose that the interaction of hsERVPRODH and PRODH may have contributed to human CNS evolution.

Functional characterization of the PRODH locus in the brain tissues. (A) Schematic view of the brain sections investigated. (B) Expression of PRODH in human brain tissues measured using qRT-PCR relative to ACTB. Data show means ± SD of three independent experiments. (C) Representative methylation patterns of the hsERVPRODH (Left) and CpGPRODH (Right) in the left hippocampus and left hemisphere of the frontal lobe. Black circle, methylated CG dinucleotide; white circle, unmethylated CG dinucleotide. (D) High-resolution melting profiling of bisulfite-treated DNA from the left (l) and right (r) hemisphere of human hippocampi. Data are shown for two PCR-amplified bisulfite-treated CG-rich fragments of the hsERVPRODH. C.meth, methylated sequence control; C non-meth, unmethylated sequence control. (E) Transcriptional activity of PRODH in human and chimpanzee brain tissues. The data were extracted from the NCBI GEO database, and the fold-change differences in gene-expression levels in individual human tissue samples and in the average chimpanzee tissue were calculated. Arbitrary units represent the fold-change difference between the PRODH expression in human samples and the chimpanzee median PRODH expression.

Transformation of rat hippocampal cells with a lentiviral construct carrying the upstream human PRODH region. (A) Schematized structure of the lentiviral construct. Red fluorescent protein (DsRed) was placed under the transcriptional control of the PRODH promoter; the GFP gene was placed under the control of the constitutive CMV promoter (+ control). (B) PRODH promoter activity in different hippocampal cell types. (C) Comparison of DsRed vs. GFP fluorescence in astrocytes (a) and neurons (b).